Based on amphiphiles, osmotic stress, and protease inhibitors, we hypothesize that egress is pressure driven through folding and fragmentation of the enzymatically altered erythrocyte membrane. Osmotic pressure could build up in either the parasitophorous vacuole (PV) or the host cell cytoplasm. We propose and test the idea that parasites easily pass through a hemoglobin-depleted erythrocyte cytoplasm and breach a weakened erythrocyte membrane. The vacuole swells several minutes before parasite egress. Starting adjacent to the parasites space, vacuole swelling later extends in all directions. At the same time, the visible area of the erythrocyte compartment shrinks, suggesting redistribution of water between the erythrocyte cytosol and vacuole. Eventually, dissociated parasites leave the host cell by breaching first the PVM (presumably when PV critical volume is reached) and then the erythrocyte membrane. Thus, dependence of egress on osmotic pressure can be described in terms of erythrocyte hydration, which affects the swelling and rupture of the vacuole. To test the relationship between erythrocyte hydration and parasite egress, we used dehydrated erythrocytes from donors homozygous for sickle hemoglobin gene (HbSS versus normal HbAA). Sickle erythrocytes do support P. falciparum replication, but dehydrated sickle cells did not allow normal egress. The decreased erythrocyte volume may contribute to malarial protection in individuals with sickle erythrocytes. Notably, decreased erythrocyte volume is a characteristic for individuals with thalassemia and iron deficiency. Alternatively, one may speculate that P. vivax gained an advantage by targeting reticulocytes, the largest circulating erythrocytes in the host. The negative effect of erythrocyte dehydration on parasite egress, demonstrated here, and on invasion emphasizes the general importance of host cell hydration for the asexual cycle of malaria parasites. In this project, we found two new essential steps in the program of Plasmodium falciparum egress from erythrocytes. First, the parasitophorous vacuole swells as the erythrocyte shrinks, suggesting ion and water redistribution between these two compartments of infected cells. At the end of the cycle, vacuole swelling apparently provides the space for parasite dissociation prior to egress, leading to vacuole rupture. In the midst of erythrocyte shrinkage, the tension of erythrocyte membrane decreases without loss of integrity. Second, parasite egress requires host cell membrane poration prior to host cell membrane rupture. Membrane poration is observed in erythrocytes that are not swollen, thus it does not result from critical membrane stretching. Perhaps either release of protein from erythrocytes or an influx of ions into the host cells, or both, is needed for the asexual parasite cycle to complete. Alternatively, host cell membrane poration could serve to weaken a barrier that parasites must breach to egress. Similarities in parasite egress mechanisms between two families of the phylum Apicomplexa, Plasmodium, and Toxo- plasma are emerging: both type of parasites make pores in host cell membrane and activate host cell calpain prior to egress. Because P. falciparum has multiple experimental limitations, Toxoplasma, a more conventional organism, emerges as a model for Apicomplexan biology. Regardless, egress of parasites is a vital step of diseases devastating humanity. Our appreciation of a more complex egress program provides more targets for novel antimalarials, just as it may help to explain the selective advantage that the sickle trait confers upon its carriers. 2) Electron tomography (ET) provides a three-dimensional (3D) view of cellular ultrastructure at nanoscale spatial resolution, and thus gives unique insight into the supramolecular basis of biological processes. In this project we investigated an alternative approach to electron tomography that yields 3D reconstructions of thicker (1m) sections at resolutions comparable to conventional ET of thin sections. The ability to perform 3D reconstructions from larger volumes is particularly attractive for studying unicellular eukaryotic microorganisms, some of which are sufficiently small to be contained within just a few serial sections. Our approach is also valuable for reconstructing entire mammalian cells using serial thick-section tomography. Scanning transmission electron tomography using a tightly focused electron probe can overcome some of the limitations imposed by conventional ET. First, the ability to focus the probe dynamically in STEM enables in-focus imaging of very large specimen areas even at the highest tilt angles. Second, because in STEM there are no image-forming lenses after the specimen, the resolution in images of thick specimens is not degraded by chromatic aberration. Generating high resolution STEM tomograms from entire cells that span several micrometers in depth can be accomplished by imaging serial 1-2 &#956;m-thick sections. Here we demonstrate the feasibility of reconstructing an entire human erythrocyte infected with important human pathogen Plasmodium falciparum, the causative agent of malaria, from only four consecutive 1 &#956;m-thick sections. Tomogram slices of one infected erythrocyte revealed the parasite during the process of schizogany, i.e. multiple nuclear divisions and formation of new parasites. At this stage of the parasite cycle active morphogenesis multiplies or produces de novo intracellular organelles for up to 32 new parasites within one schizont. The dynamics of morphogenesis is poorly understood because of the laborious procedure of 3D reconstructions of the serial thin sectioning of cells with complex architecture. The spatial resolution achievable with the new method allows us to identify the major organelles of schizont in thick sections, such as nuclei, rhoptries, pigment vacuole, rough endoplasmic reticulum, Golgi complex, apicoplast, and lipid body. Three layers of membranes surrounding the schizont are clearly identifiable: 1) the parasite plasma membrane, caught at the onset of invagination to form new parasites, 2) the membrane of the parasitophorus vacuole and 3) the erythrocyte membrane. Parasite-derived membrane structures such as tubular extensions of the vacuolar membrane, Maurers clefts and circular clefts, are visible inside erythrocyte cytoplasm as well. Thus, a new ultrastructural method is now available to study the complex dynamics of malaria parasite development inside human erythrocyte. The significance of this technique is that 10 nm resolution is sufficient to discern organelles, and the process of organellogenesis during schizogeny is currently obscured by the amorphous nature of standard ultrastructural views. This new technique is rapid enough to allow a series of schizonts to be studied and the morphological sequence of events established, as was recently achieved for parasite release. Already new objects are emerging whose identity is unclear and will require elaboration of labeling techniques for markers. Thus STEM tomography using axial detection for imaging thick sections of biological specimens at a spatial resolution around 5-10 nm, which is comparable to the spatial resolution of traditional ET from thin sections (typically 3-8 nm) is both feasible and advantageous. The demand for high-resolution, large-volume imaging of biological specimens has been answered so far by the large scale application of traditional ET of thin sections. The present study suggests that it will be possible to reconstruct conveniently and efficiently entire mammalian cells through serial thick-section STEM tomography.